INFECTlON AND IMMUNITY, Mar. 1994, p. 818-827 Vol. 62, No.

3
0019-9567/94/$04.00+0
Copyright 1994, American Society for Microbiology

CD4+ T-Cell Clones Obtained from Cattle Chronically Infected

with Fasciola hepatica and Specific for Adult Worm Antigen
Express Both Unrestricted and Th2 Cytokine Profiles
WENDY C. BROWN,I* WILLIAM C. DAVIS,2 DIRK A. E. DOBBELAERE,3
AND ALLISON C. RICE-FICHT4
Department of Veterinary Pathobiology' and Department of Medical Biochemistry and Genetics,4 Texas A&M
University, College Station, Texas 778431; Department of Veterinary Microbiology and Pathology, Washington State
University, Pullman, Washington 991642; and Institute of Parasitology, University of Bern, Bern, Switzerland3
Received 9 September 1993/Returned for modification 5 November 1993/Accepted 16 December 1993

The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many
experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola
hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were
characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult
worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and -y/8 T-cell-receptor-
bearing T cells. However, cell lines containing either fewer than 10%0v CD8+ T cells or depleted of 'y/8 T cells
proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for
proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine
expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4,
gamma interferon (IFN-y), and tumor necrosis factor and Northern (RNA) blot analysis to verify the
expression of IL-2, IL-4, and IFN-,y revealed that the Th-cell clones expressed a spectrum of cytokine profiles.
Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-'y
mRNA. The majority of Th-cell clones were classified as ThO cells by the expression of either all three cytokines
or combinations of IL-2 and IL-4 or IL-4 and IFN-,y. No Thl-cell clones were obtained. All of the Th-cell clones
expressed a typical memory cell surface phenotype, characterized as CD45RIW, and all expressed the lymph
node homing receptor (L selectin). These results are the first to describe cytokine responses of F.
hepatica-specific T cells obtained from infected cattle and extend our previous analysis of ThO and Thl cells
from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect.
Immun. 61:3273-3281, 1993) to include F. hepatica-specific Th2 cells.

The digenetic trematode parasite Fasciola hepatica is a
commonly occurring liver fluke that infects a wide variety of
mammals, including cattle, sheep, and humans. Fascioliasis can
be a chronic disease, with adult worms surviving for up to 12
years in the host. The major pathology caused by F. hepatica
includes fibrosis due to severe trauma induced by juvenile
worms migrating through the liver and inflammation, edema,
and fibrosis of the bile ducts due to the presence of the adult
flukes. Acquired immunity to this parasite differs in different
species (reviewed in reference 22). Whereas sheep and rabbits
do not appear to acquire resistance to infection by F. hepatica,
cattle, goats, and rats exhibit substantial immunity to challenge
infection with F. hepatica when they have had a previous
infection. In cattle, it was shown that worm rejection occurred
24 weeks after a primary infection (18), leading to resistance to
a secondary infection characterized by a decrease in the size
and number of recovered flukes.
Protective immunity can be elicited in cattle, goats, and rats
by infection with irradiated metacercariae, somatic fluke ex-
tracts, and excretory/secretory fluke antigens (22). More recent
studies have indicated the feasibility of achieving partial pro-
tective immunity in cattle and sheep vaccinated with purified
or recombinant proteins of F. hepatica, including a 12-kDa
*
Corresponding author. Mailing address: Department of Veteri-
nary Pathobiology, Texas A&M University, College Station, TX
77843-4467. Phone: 409-845-4207. Fax: 409-845-9972. Electronic mail
address: wbrown@vthvax.tamu.edu.
818
antigen (24) and a 26-kDa protein, glutathione S-transferase
(46). However, in spite of these accomplishments, the nature
of the protective immune response to F. hepatica has not been
clearly defined. Both humoral and cell-mediated mechanisms
of immunity appear to be important for resistance to F.
hepatica infection in different species (22). However, the
following observations suggest that antibody alone is not
sufficient for protective immunity. First, passive transfer of
resistance in rats by immune serum was achieved only with
large volumes (1), and an insignificant level of immunity was
achieved in a study involving the transfer of immune serum
from one calf to its naive twin (13). Second, serum antibody
titers of infected rats did not correlate with the number of
flukes recovered at necropsy (30). Finally, resistance in sheep
vaccinated with F. hepatica glutathione S-transferase did not
correlate with antibody titers (46).
In mice and humans, helminth infections are generally
characterized by high levels of immunoglobulin E (IgE) and
eosinophils, induced by interleukin-4 (IL-4) and IL-5. These
cytokines are produced by the helper T2 (Th2) subset of CD4+
T cells (20). However, protective immunity in experimental
murine schistosomiasis caused by Schistosoma mansoni ap-
pears to be dependent on the Thl subset of T cells that
produce IL-2 and gamma interferon (IFN--y) (10, 48). Protec-
tive immunity is apparently effected by IFN-y-activated mac-
rophages that, in vitro, are able to kill schistosomula (28). In
addition, downregulation of Thl-cell responses by Th2-cell-
derived IL-10 in mice undergoing egg-laying infections appears
VOL. 62, 1994 F. HEPATICA-SPECIFIC ThO AND Th2 CELLS IN CATTLE
to play an important role in the chronicity of infection (47). In
contrast to the mouse model, studies performed with the rat
model of S. mansoni have strongly suggested a major role for
Th2 cells in protective immunity through the stimulation of
IgE, eosinophils, and mast cells (11). In this model, cellular
immunity is mediated by eosinophils and macrophages, which
in the presence of specific antibody including IgE, are activated
to kill schistosomula in vitro. It thus appears that the cytokine-
dependent effector mechanisms against S. mansoni and F.
hepatica may differ in different host species, underscoring the
need to define the mechanisms of immunity against these
parasites in the natural host.
The importance of T cells in protective immunity against F.
hepatica was indicated by studies performed with rats and
cattle, which demonstrated successful transfer of resistance
with spleen and lymph node cells from infected donors to
syngeneic recipients (1, 13, 40). However, few studies have
attempted to characterize T-lymphocyte responses in cattle
against F. hepatica. In vitro proliferation assays revealed early
and transient responses of peripheral blood mononuclear cells
(PBMC) against F. hepatica adult worm antigen, which disap-
peared by 5 weeks postinfection (37). Furthermore, skin tests
performed after 4 weeks postinfection failed to demonstrate
delayed-type hypersensitivity in fluke-infected cattle (19). The
reasons for the discrepancy between the ability to transfer
resistance with lymphoid cells and the inability to detect
cell-mediated immune responses by S weeks following experi-
mental infection are unclear. One possibility is that by 4 to 5
weeks postinfection, cell-mediated immune responses, effected
by Thl-like cells, are downregulated by IL-10 produced in
response to antigens released by migrating worms. Clearly, a
detailed investigation of the molecular mechanisms of immu-
nity to F. hepatica is needed before rational vaccine strategies
can be devised.
The importance of cytokines in response to different hel-
minth infections, including the related trematode parasite S.
mansoni, and the need to understand the molecular basis of
immunity in fascioliasis in the natural host prompted a detailed
investigation of the bovine T-cell response against F. hepatica.
Since polyclonal lymphoid-cell responses are often unreliable
because of complex cellular interactions, parasite-specific T-
cell lines and T-cell clones were derived from PBMC of
infected cattle for phenotypic and functional characterization
of the F. hepatica-specific T-cell response. Adult worm extract
was selected as an antigen for these studies, since this prepa-
ration was capable of stimulating protective immunity in cattle
(22). We found that bovine Th-cell clones specific for helminth
antigens expressed both unrestricted and Th2 cytokine profiles,
as reported for Th-cell clones derived from humans and mice
(12, 17, 20, 50). The experiments described in the present study
are the first to characterize the bovine T-cell response against
F. hepatica as well as Th2 cells in cattle and provide a basis for
further studies of the immune response in cattle resistant to
challenge infection with F. hepatica. T-cell clones may be
useful for subsequent identification of potentially protective
antigens of this parasite.
MATERIALS AND METHODS
Experimental infection and serological responses. Two 15-
month-old Charolais cattle (one heifer, designated animal Gl,
and one steer, designated animal G8) were purchased from
Granada Biosciences, Inc., Houston, Tex. These animals were
produced by embryo cloning and were genetically identical to
heifer G3 and steer G6, respectively, which were infected and
challenged with Babesia bovis as described in an earlier publi-
819
cation (7). Cattle Gl and G8 were infected per os with 1,000
metacercariae of the Oregon strain of F. hepatica (Baldwin
Aquatics, Monmouth, Oreg.). Serum samples obtained prior to
and at 3- to 10-month intervals following infection were
obtained for use in immunoblot assays. Sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis and immunoblotting
were performed essentially as described elsewhere (6) and
employed the minigel and transblot systems (Bio-Rad Labora-
tories, Richmond, Calif.), with 10% acrylamide gels and 30 ,ug
of F. hepatica worm antigen per lane. Molecular mass stan-
dards obtained from Bio-Rad included 3-galactosidase (116
kDa), rabbit muscle phosphorylase b (97 kDa), bovine serum
albumin (66 kDa), hen egg white ovalbumin (45 kDa), and
bovine carbonic anhydrase (31 kDa). Fractionated proteins
were electrophoretically transferred from acrylamide to nitro-
cellulose filters for 1 h at 100 V. Preinfection and postinfection
bovine sera were used to develop the immunoblots. Sera were
diluted 1:100 in Tris-buffered saline (10 mM Tris, 150 mM
NaCl [pH 7.5]) containing 1% gelatin, and serologically reac-
tive proteins were detected with a 1:5,000 dilution of alkaline
phosphatase-conjugated goat anti-bovine IgG (Kirkegaard &
Perry Laboratories, Gaithersburg, Md.) and the chromogenic
substrates nitroblue tetrazolium and 5-bromo-4-chloro-3-in-
dolyl-phosphate (Sigma Chemical Co., St. Louis, Mo.) as
instructed by the manufacturers.
Parasite antigens. Adult F. hepatica flukes were obtained
from infected bovine livers collected at a local abbatoire
(Braunfels Meat, Inc., Sealy, Tex.). Flukes were teased from
the bile ducts after transverse slices were made across the
entire ventral surface of the liver. Flukes were washed three
times in 0.15 M phosphate-buffered saline (PBS), pH 7.4,
frozen, and stored at -70C until use. To prepare the adult
worm antigen extract, 15 frozen adult flukes were ground to a
fine powder and suspended in 10 ml of PBS in the presence of
2 mM phenylmethylsulfonyl fluoride and 5 mM iodoacetamide.
The suspension was homogenized with a tissue homogenizer
and centrifuged at 15,000 x g at 4C for 1 h. The supernatant
was diluted to a concentration of 1 mg of protein per ml and
sterilized by filtration through a 0.22-,um-pore-size filter. For
lymphocyte proliferation assays, the antigen was prepared as
described, except that iodoacetamide was not included. A
crude membrane preparation of B. bovis merozoites was
prepared from the Mexico isolate as described elsewhere (6)
and used as a control antigen for lymphocyte proliferation
studies. The protein contents in both parasite antigenic prep-
arations were determined by the Bradford protein assay (Bio-
Rad) with bovine IgG as a protein standard. Aliquots of the
different antigens were stored at - 70C.
Generation of F. hepatica-specific T-cell lines and clones.
T-cell lines specific for F. hepatica adult worm antigen were
established from PBMC of infected cattle Gl and G8 essen-
tially as described for T-cell lines specific for B. bovis (2, 5). In
brief, PBMC were obtained from infected animals at 27 weeks
(cell lines G1.5 and G8.5), 34 weeks (cell lines G1.6 and G8.6),
and 40 weeks (G8.7) following infection. PBMC were stimu-
lated in 1.5-ml cultures in 24-well plates (Costar, Cambridge,
Mass.) at a density of 4 x 106 PBMC per well with 25 or 50 ,ug
of adult worm antigen per ml of complete RPMI 1640 medium
(6). After 7 days, viable cells were subcultured to a density of
5 x 105 cells per well and were restimulated with antigen and
2 x 106 irradiated (3,000 rads) autologous PBMC as a source
of antigen-presenting cells (APC).
Cell lines G1.6 and G8.6, which had been cryopreserved
after 1 week of culture and thawed, were treated with a
monoclonal antibody (MAb) specific for the bovine -y/8 T-cell
receptor complex, designated TcR1-N12 (14), and rabbit com-
820 BROWN ET AL.
plement to remove y/8 T cells. Briefly, 107 T cells were
incubated for 30 min at 4C with MAb CACT-61A diluted in
PBS to a final concentration of 15 ,ug of protein per ml. The
cells were washed once in PBS and were then incubated for 30
min at 37C with an equal volume of rabbit complement
(Sigma) diluted 1:8 in PBS. The cells were then washed three
times in complete medium and stimulated with antigen and
APC as described. These cell lines were designated G1.6Rx
and G8.6Rx. Cell line G8.7Rx was derived from freshly iso-
lated PBMC that were subjected to two consecutive treatments
with MAb CACT.61A and complement and was cultured with
F. hepatica antigen as described.
Cell lines were maintained in culture for up to 7 weeks by
weekly stimulation with antigen and APC and were assessed
for antigen-specific proliferation or the presence of surface
markers 7 or 8 days following the last stimulation with antigen
and APC. Lines G1.6Rx, G8.6Rx, and G8.7Rx were cloned by
limiting the dilution after 2.5 weeks of culture. Cloning was
performed in 96-well round-bottom plates (Costar) essentially
as described elsewhere (8), with the following modifications. A
statistical average of 1 or 0.3 cells per well was stimulated with
25 ,ug of F. hepatica adult worm antigen per ml of complete
medium containing 10% bovine T-cell growth factor (TCGF)
(3) and 5 x 104 irradiated (3,000 rads) autologous PBMC.
Proliferating cells were transferred successively to 48-well
(Costar) and 24-well plates, stimulated with antigen, TCGF,
and APC, and tested for antigen-dependent proliferation.
Cloning frequencies of 60 to 75% and 15 to 25%, respectively,
were obtained from the cell lines when an average of 1.0 or 0.3
T cells was distributed per well.
Cell surface phenotypic analysis. PBMC, F. hepatica-specific
cell lines, and T-cell clones were stained with a panel of MAbs
by indirect immunofluorescence as described elsewhere (5)
and were analyzed with a Coulter EPICS 741 flow cytometer.
MAbs specific for bovine leukocyte surface markers obtained
from the International Laboratory for Research on Animal
Diseases, Nairobi, Kenya, included IL-A51 (specific for CD8),
IL-A12 (specific for CD4), IL-A26 (specific for CD2), and
IL-A150 (considered specific for a polymorphic determinant
on bovine CD45RO) (32, 33). MAbs obtained from Chris
Howard at the Agriculture and Food Research Council Insti-
tute for Animal Health in Compton, United Kingdom, in-
cluded CC76 (specific for bovine CD45R; 26) and CC32
(specific for bovine L selectin; 25). MAb CACT-61A (specific
for the -y/8 TcR1-N12 determinant; 14) was used to enumerate
y/8 T cells in PBMC and T-cell lines. Goat anti-mouse
immunoglobulin [affinity-purified F(ab')2 fragments; Cappel/
Organon-Teknika, Inc., Malvern, Pa.] was used as a second
reagent. The percentages of positive cells were determined by
subtracting the percentages of cells stained with the second
reagent only from the percentages of cells stained with a given
MAb and the second reagent, as described by Overton (38).
The proportion of CD4+, CD8+, and ry/8 T cells was deter-
mined by the following equation:
100 x
% cells stained with a given MAb
% IL-A12+ cells + % IL-A51+ cells + % CACT.61A+ cells
Lymphocyte proliferation assays. Proliferation assays were
carried out in duplicate wells of half-area 96-well plates
(Costar) at 37C in a humidified atmosphere of 5% CO2 in air
for 3 days as described elsewhere (5). Each well (total volume,
100 [lI) contained complete medium, with responder cells
added at a final concentration of 3 x 105 cells per ml (obtained
7 days following the last stimulation of the T-cell lines or clones
with antigen) and 2 x 106 autologous or allogeneic (animal
INFECT. IMMUN.
C97), irradiated PBMC per ml as a source of APC. Antigens
were added to the assay mixes at a final concentration of 1 to
50 ,ug of protein per ml of complete medium. Bovine TCGF (4
or 5%) was included as a positive control for lymphocyte
proliferation. In some experiments, clones G8.3B7 and
G1.2H4 were assayed in the presence of 5% TCGF, in addition
to antigen, in all wells of the proliferation assays. Proliferation
was determined by measuring the incorporation of 0.25 ,uCi of
[I25I]iododeoxyuridine (125IUDR; ICN Radiochemicals, Inc.,
Costa Mesa, Calif.) added during the final 4 h of the assay. The
cells were harvested, and the radioactivity was counted in a
gamma counter. The results are presented as the mean counts
per minute and standard deviations of duplicate samples.
The one-tailed Student t test was used to determine the
levels of significance between control and experimental cul-
tures.
Stimulation of cells and biological assays for cytokine
production. T cells obtained 6 or 7 days after the last stimula-
tion with antigen and APC were washed in complete medium
and cultured for 17 to 18 h at a concentration of 1.3 x 106 cells
per ml of complete medium containing 5 pug of concanavalin A
(ConA; Sigma) per ml in the absence of APC. In one experi-
ment, F. hepatica-specific T-cell clone G1.3E11 was cultured
with either ConA or with 25 ,ug of antigen per ml and APC for
18 h, and supernatants were compared. Controls included
ConA diluted to 5 ,ug per ml of complete medium and
supernatants collected from APC cultured with antigen in the
absence of T cells. Supernatants were harvested by centrifuga-
tion and stored at - 80C.
Biological assays for cytokines have been described in detail
elsewhere (7-9). In brief, IFN--y activity was measured in a
microtiter cytopathic-effect assay with vesicular stomatitis virus
and Madin-Darby bovine kidney (MDBK) cells. MDBK cell
survival was evaluated by the 4-h uptake of 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye in
the cells. IFN-y titers were compared with a human recombi-
nant IFN-ax2 reference reagent (National Institute of Allergy
and Infectious Diseases, Bethesda, Md.; catalog number,
GxaOl-901-535). For determination of tumor necrosis factor
(TNF) activity in the supernatants of T-cell clones, a TNF-
sensitive WEHI-164 subline was incubated with culture super-
natants in a 48-h assay, and cytopathicity was determined by
the MTT dye reduction assay. TNF titers were compared with
a standard human recombinant TNF-ct (Upstate Biotechnol-
ogy, Inc., Lake Placid, N.Y.). A cloned CD8+, IL-2-dependent
bovine T-cell line, designated 99.G1.G3, was used to measure
IL-2 or IL-4 activity. The cell line did not discriminate IL-4
from IL-2 activity, since it expressed both IL-2 and IL-4
mRNA and proliferated upon ConA activation (7). Delecti-
nated culture supernatants of F. hepatica-specific T-cell clones
stimulated with ConA and supernatant from antigen-stimu-
lated clone G1.3E11 were diluted 1:2 to 1:256 in complete
medium and added to duplicate wells of 99.G1.G3 cells which
had been distributed at a density of 3 x 104 cells per well in
96-well half-area microtiter plates. In all assays, recombinant
human IL-2 (Boehringer Mannheim, Indianapolis, Ind.) was
serially diluted from 100 to <1 U/ml for use as a reference
standard. Cells were cultured for 48 to 72 h, radiolabeled,
harvested, and counted as described above. A semiquantitative
estimate of IL-2 and IL-4 activity in the culture supernatants
was obtained by comparison with a standard curve of human
IL-2.
Northern (RNA) blot analysis. Analysis of mRNA expres-
sion of IL-2, IL-4, and IFN--y was performed by Northern
blotting as described elsewhere (7, 9). Total RNA was obtained
from unstimulated bovine turbinate (BT) cells as a negative
Vo[-. 62, 1994 F. HEPATICA-SPECIFIC ThO AND Th2 CELLS IN CATTLE 821

TABLE 1. Response of F. hepatica-specific T-cell lines to

Gl G8 F. hepatica adult worm antigen
A B C D A B C D Radioactivity (mean cpm + SD) incorporated by
1~ ~ G Cell line and no. of T-cell lines stimulated with the following antigens":
wks in culture None
116- - _-
(medium) B. boOis F. hepatica
66 - _I

G1.5
2 434 36 414 76 34,894 87
45
7 2,936 66 706 48 14,418 99
G8.5
2 328 64 325 26 29,391 254
7 2,332 26 1,796 41 5,341 271
G1.6
1 203 65 315 20 18,074 5,915
3 2,902 341 2,197 + 116 24,851 1,780
FIG. 1. Serologic reactivity of serum samples from F. hepatica- Gl.6Rx (y/8 T-cell
infected cattle with adult worm antigen. Western blots were performed depleted)"
by using 30) ,ug of F. lhepatica adult worm antigen per lane and 5 396 78 519 74 88,783 217
preimmune sera from animals GI and G8 (lane A) and sera obtained G8.6
3 months (lane B), 5 months (lane C), or 10 months (lane D) following 1 243 37 263 45 5,245 931
oral infection of cattle G1 and G8 with 1,000 metacercariae. Molecular 3 503 155 238 3 31,121 1,875
size standards are indicated on the left and right of the figure. G8.6Rx (y/8 T-cell
depleted)"
5 1,382 266 588 106 83,349 1,962
control, from PBMC stimulated for 18 h with ConA as a
positive control, and from T-cell clones that had been cultured " Results are presented for 25 ,ug of B. bovis merozoite membrane antigen and
for 8 h with ConA, by using 2 ml of RNazol B (Biotecx 51) ,ug of F. hepatica adult worm antigen per ml. The results of proliferative
Laboratories, Inc., Houston, Tex.) per 107 cells, as specified by responses of cell lines cultured with F. hepatica antigen were shown to be
significantly different from responses of cell lines cultured with either complete
the manufacturer. Total RNA was also obtained from T-cell medium alone or B. bovis (P < 11.01) by the Student one-tailed t test.
clones stimulated for 13 h with 50 ,ug of adult worm antigen per "'Cell lines G 1.6 and G8.6 were cryopreserved after 1 week of culture, thawed,
ml and APC or from control APC cultured in the absence of T treated with MAb CACT.61A and complement. and cultured for an additional 4
cells. RNA (15 to 20 pg) was subjected to electrophoresis in a weeks. The y/8 T-cell-depleted lines are designated GI.6Rx and G8.6Rx.
formaldehyde-morpholinepropanesulfonic acid (MOPS)-
1.6% agarose gel, transferred to GeneScreen nylon filters
(Dupont NEN, Boston, Mass.), and hybridized with the fol- ous bands ranging in apparent molecular mass from approxi-
lowing cDNA probes. The IL-2 cDNA probe consisted of a mately 26 to >116 kDa. However, during the course of
0.79-kb EcoRI fragment of the bovine IL-2 cDNA (41) and was infection, the pattern of reactivity with F. hepatica varied. For
a generous gift from Raymond Reeves, Washington State
example, 5 months postinfection serum from animal GI (lane
University. The IL-4 cDNA probe consisted of a 0.4-kb C) displayed decreased reactivity with bands ranging from 60
EcoRI-SalI fragment of bovine IL-4 cDNA (23). The IFN-y to 120 kDa, compared with 3-month (lane B) and 10-month,
probe consisted of a 0.5-kb XbaI-DraI fragment of bovine (lane D) postinfection sera, whereas 5-month postinfection
IFN--y cDNA and was kindly provided by Arjun Singh, Genen- serum from animal G8 (lane C) recognized additional high-
tech, San Francisco, Calif. The actin cDNA probe consisted of molecular-mass bands of -116 kDa that were less visible in
a 1.0-kb EcoRI fragment of bovine actin cDNA (15) and was
3-month (lane B) and 10-month (lane D) postinfection serum
kindly provided by Angelika Ehrfeld, Max Planck Institute for samples. In addition, 10 months postinfection, serum from
Immunology, Freiburg, Germany. The cDNAs were labeled animal GI recognized a novel band of <26 kDa. These results
with 39P by the random primer method with a kit from show that at the time at which the T-cell lines were established
Boehringer Mannheim, yielding probes with specific activities from these animals (approximately 7 to 10 months postinfec-
of 1 x 10'9 to 2 x 109) cpm/p.g. Filters were prehybridized and tion), serologic reactivity, indicative of an active or recent
hybridized with radiolabeled probes (7, 9) and were then infection, was still present.
exposed to HyperfilmMp (Amersham Corporation, Arlington F. hepatica-specific T-cell lines. To characterize the nature
Heights, Ill.) at 80C. The approximate sizes of the tran-
-
of the T-cell response to F. hepatica adult worm antigens, F.
scripts in kilobases were determined from nucleic acid molec- hepatica-specific T-cell lines were established from PBMC
ular size standards (0.24- to 9.5-kb RNA ladder; GIBCO-BRL, obtained from cattle infected 7 to 10 months earlier. All cell
Gaithersburg, Md.) that were electrophoresed, transferred to lines responded to either 10 or 50 p.g of F. hepatica adult worm
nylon, and stained with methylene blue. antigen per ml of medium (Table 1 and data not presented)
but did not respond to any concentration of control antigens,
RESULTS including membrane antigen prepared from either B. bovis
merozoites (Table 1) or uninfected erythrocytes (data not
Serologic responses of F. hepatica-infected cattle. Serum presented). Cell lines G1.5 and G8.5 were cloned after 3 weeks
samples obtained from animals GI and G8 before infection of culture, and 10 T-cell clones were obtained. None of the 10
and at 3, 5, and 10 months following oral infection with 1,000 clones proliferated in response to F. hepatica adult worm
metacercariae of F. hepatica were compared for reactivity with antigen, and all of the clones expressed the y/8 TcR (data not
adult worm antigenic extract. Figure shows that, compared presented). This finding prompted a careful evaluation of the
with preinfection serum samples (lane A), postinfection serum relative proportions of CD4+, CD8+, and y/8 TcR+ T cells in
samples (lanes B to D) reacted on immunoblots, with numer- the cell lines stimulated with F. hepatica. In the majority of F.
822 BROWN ET AL. INFECT. IMMUN.

TABLE 2. Cell surface phenotypic analysis of F. hepatica-specific TABLE 3. Response of T-cell clones from cattle GI and G8 to
T-cell lines F. hepatica adult worm antigen
Percentage of the total T-cell population Radioactivity (mean cpm SD) incorporated by F. hepatica-
Cell line and no. of stained by a MAb' T-cell specific T-cell clones stimulated with the following antigens":
wks in culture IL-A12 IL-A51 CACT.61A clone F. hepatica +
Medium TCGF F. hepatica TCGF
(CD4) (CD8) (-y/8 TcR1)
G1.5 G8.2G10 16 4 6,068 160 46,668 + 6,626 56,751 3,244
0 (PBMC) 51.1 25.9 23.0 G8.3B7 23 22 340 26 9,627 110 49,811 1,795
6 22.4 27.7 49.9 GI.3G10 6 6 879 136 10,340 1,410 70,500 1,348
G8.5 G1.3G4 7 5 238 17 22,923 183 84,912 + 1,630
0 (PBMC) 41.3 28.5 30.2 G1.3E11 24 1 7,551 + 513 43,001 + 745 82,990 + 4,626
6 22.9 26.2 50.9 GI.3B11 13 6 2,724 + 11 13,877 + 133 38,216 + 189
G1 .6 Gl.lH5 44 3 15,627 1,133 32,545 59 59,011 4,079
1 55.6 14.1 30.4 GI.1H12 51 2 1,081 + 94 59,873 256 83,906 4,687
3 35.6 15.4 48.9 G1.2H4 8 6 2,240 + 30 42 2 23,278 + 471
G1.6Rx (y/8 T-cell G8.2C12 27 9 1,218 191 19,991 + 1,955 44,865 94
depleted)' G8.2C1 24 8 8,802 633 56,144 + 2,118 81,887 + 618
5 85.5 6.3 8.4 G8.2C3 25 2 428 11 44,638 1,238 41,060 434
G8.6 G8.2B1A 28 3 4,102 + 312 90,940 + 3,210 102,610 + 1,345
1 35.5 27.6 36.9 G8.2D9 13 1 6,808 192 18,054 1,397 27,767 + 756
3 34.8 7.8 57.4 G1.3G5 16 4 687 37 1,597 371 43,269 1,887
G8.6Rx (y/8 T-cell G1.3F7 25 9 300 24 475 47 7,117 887
depleted)' G1.2B6 67 6 3,658 234 5,676 + 144 20,211 + 1,111
5 88.9 2.2 8.8
" Antigen consisted of 50 p.g of F. hepatica adult worm antigen per ml. TCGF
" Freshly isolated PBMC or T cells obtained from cell lines cultured for 1 to was used at a final concentration of 4%. Proliferative responses of T-cell clones
6 weeks with F. hepatica worm antigen were stained with the indicated MAb. The cultured with F. hepatica antigen were shown to be significantly different from
proportion of CD4+, CD8+, and -y/8 T cells was determined as described in the responses of T cells cultured with medium alone (P < 0.01), and proliferative
Materials and Methods section. responses of T-cell clones cultured with antigen and TCGF were shown to be
b Cell lines G1.6Rx and G8.6Rx are described in footnote a to Table 1. significantly different from proliferative responses of T-cell clones cultured with
TCGF alone (P < 0.01) by the Student one-tailed t test.

hepatica-specific T-cell lines examined, the proportion of
CD4+ T cells increased during a 4-week culture period,
whereas the proportion of CD8+ T cells decreased during this
time (data not presented), and cell lines containing fewer than
10% CD8+ T cells proliferated well to antigen (Table 2).
However, /yI8 T cells constituted between 15 and 40% of the
total T-cell population in T-cell lines stimulated with antigen
for 2 to 4 weeks and reached levels of 50 and 51% of the T cells
in lines G1.5 and G8.5, respectively, by 6 weeks of culture
(Table 2). The high percentage of y/8 T cells observed in
several of these cell lines could be partially attributed to the
relatively high numbers of -y/8 T cells in the PBMC of these
young cattle. When sampled four times over a 2-month period,
the percentages of y/8 T cells, expressed as percentages of the
total T-cell population, averaged 18.3% 2.9% for animal Gl
and 30.0% 3.6% for animal G8. Because the -y/8 T cells were
possibly interfering with the ability to clone antigen-responsive
CD4+ T cells from the lines, the y/8 T cells were lysed with
specific antibody and complement. This treatment was gener-
ally effective at removing the majority of -y/8 T cells from T-cell
lines and PBMC. The -y/8 T cell-depleted cell lines exhibited
strong proliferative responses against antigen, as shown for cell
lines G1.6Rx and G8.6Rx (Tables 1 and 2). The panel of T-cell
clones described here was derived from T-cell lines depleted of
-y/8 T cells.
Antigen-specific responses of F. hepatica-specific T-cell
clones. To obtain T-cell clones useful for future identification
of stimulatory parasite antigens and to characterize cytokines
produced by F. hepatica-specific T cells, several F. hepatica-
specific cell lines were cloned by limiting dilution and screened
for antigen-specific proliferative responses. Although the
clonal nature of the cells cannot be conclusively confirmed, the
majority of Th-cell clones were obtained from wells with
cloning frequencies of .25%, which is consistent with the
distribution of a statistical average of .1 cell per well. Fur-
thermore, the Th-cell clones have retained their antigen re-
sponsiveness over a period of several months in culture.
Twenty-two T-cell clones that responded specifically and in a
dose-dependent manner to F. hepatica adult worm antigen
were obtained. All clones proliferated in response to 10 and 50
,ug of antigen per ml of medium, with optimal responses
achieved with the highest antigen concentration (17 clones are
represented in Table 3). One clone, G1.2H4, responded to
antigen only in the presence of TCGF, although the majority of
clones were capable of proliferating in response to antigen in
the absence of exogenous growth factor. None of the clones
responded to B. bovis merozoite antigen, and none responded
to a recombinant 66-kDa fibrous tegument protein of F.
hepatica produced as a fusion polypeptide with Schistosoma
japonicum glutathione S-transferase (data not presented). All
of the clones expressed the surface phenotype characteristic of
Th cells, i.e., CD2+ CD4+ CD8- y/8 TcR1- (data not
presented; see Fig. 3).
To rule out the possibility that the proliferative responses of
the Th-cell clones were due to nonspecific mitogens or super-
antigens present in the crude worm extract, proliferation assays
were performed with APC obtained from either autologous or
allogeneic donor cattle. APC from animal C97 were previously
shown to be unable to present antigen to T-cell lines from
animals Gl and G8. The proliferative response to F. hepatica
antigen was nearly or completely abrogated in the presence of
allogeneic APC, whereas the proliferative response to TCGF
was either enhanced or only marginally inhibited when alloge-
neic APC were present in the cultures (data not presented).
Thus, the CD4+ T-cell clones appear to be major histocom-
patibility complex restricted.
Analysis of cytokines produced by F. hepatica-specific Th-
cell clones. Previous studies have indicated that maximal IL-2,
IL-4, IFN--y and TNF activities were detected in supernatants
of T cells stimulated with ConA for 18 to 22 h compared with
VOL. 62, 1994 F. HEPATICA-SPECIFIC ThO AND Th2 CELLS IN CATTLE 823

TABLE 4. Cytokine production by F. hepatica-specific CD4+

T cell clones
kB _ _
c0 9' ' _
.X . .c
0
Cytokine activity (U/ml)'
T-cell clone 1. CO CILCDW
Cc o
IL-2/IL-4 IFN-y TNF
G8.2G10 1 128 <6
G8.3B7 6 128 12
G1.3G10 1 8 <6 .6 - IL-4
G1.3G4 1 64 6
G1.3E11 4 128 2 1.4
G1.3B11 2 128 <6 IFN-Y
G1.lH5 1 64 6
Gl.lH12 6 128 12
G1.2H4 1 <4 6 2.1 Actin
G8.2C12 6 <2 <2
G8.2C1 26 32 12
G8.2C3 26 128 6
G8.2B1A 26 8 12
G8.2D9 3 192 <2 CM'
cr ()

G1.3G5 0 128 2 kB '

G1.3F7 0 24 <2 mo c6 co co co cD
G1.2B6 1 12 <2
IL-2
a Cytokine activity was measured in the supernatants of Th-cell clones cultured
for 17 to 18 h with 5 jig of ConA per ml.
.6 - fw * IL-4

8 h (7). An additional comparison revealed higher levels of all 1 .4 _0Awi, 2 IFN-y

three cytokines in supernatants of T cells stimulated with
mitogen compared with specific antigen (B. bovis) and APC
(7). Analysis of cytokines secreted by F. hepatica-specific clone
G1.3E11 similarly revealed that higher supernatant levels of
IFN--y and IL-2 or IL-4 activities were detected when ConA
was used as a stimulus. T cells stimulated for 18 h with 50 ,ug
of F. hepatica adult worm antigen per ml of medium and APC
secreted 32 U of IFN--y, 2 U of IL-2 or IL-4, and 2 U of TNF
per ml, whereas cells stimulated with ConA secreted 128 U of
IFN--y, 4 U of IL-2 or IL-4, and 2 U of TNF per ml. Control
supernatants of APC cultured with antigen and medium
supplemented with ConA did not contain detectable levels of
any cytokine. Because several studies have reported that the
same cytokines are induced in T cells stimulated with either
ConA or antigen and APC (21, 29, 35, 44), and since ConA
induced quantitatively higher levels of cytokines compared
with antigen and APC in bovine T-cell clones, this mitogen was
used to induce cytokines in the panel of F. hepatica-specific
T-cell clones described in the present study (Table 4). In
general, the levels of IL-2 or IL-4 activity in the supernatants
were low (s6 U/ml), with the exception of clones G8.2C1,
G8.2C3, and G8.2B1A, which produced 26 U of IL-2 or IL-4
per ml. IFN--y titers ranged from <2 to 192 U/ml. TNF titers in
the supernatants ranged from undetectable to 12 U/ml.
Because neutralizing antibodies directed against bovine IL-2
and IL-4 are not available for use in distinguishing IL-2 and
IL-4 activities detected by our bioassay, Northern blotting was
performed to identify cytokine mRNAs expressed by the panel
of F. hepatica-specific T-cell clones. In addition to hybridizing
mRNA with cDNA probes for bovine IL-2, IL-4, and IFN--y, a
bovine actin cDNA probe was used to verify the relative
quantities of RNAs on the filters. RNA extracted from PBMC
stimulated for 18 h with ConA was used as a positive control,
and RNA extracted from unstimulated BT cells was used as a
negative control for cytokine mRNA expression in the hybrid-
ization studies. We had previously determined that expression
of IL-2, IL-4, and IFN-y mRNA in activated Th-cell clones was
much stronger at 8 h than at 18 to 24 h poststimulation (7, 9),
and similar kinetics of cytokine mRNA expression were ob-
2.1 - Actin
FIG. 2. Northern blot analysis of RNA from CD4+ F. hepatica-
specific T-cell clones from infected animals Gl and G8. As a positive
control, RNA was prepared from ConA-stimulated PBMC (ConA BI),
and as a negative control, RNA was prepared from BT cells. A 20-jig
amount (or 15 jig for clones G8.2C12 and G8.2C1) of total cellular
RNA was electrophoresed on agarose gels, transferred to nylon
membranes, and probed with the indicated cytokine probes, including
actin as a control for semiquantification of RNA. Filters were exposed
for 4 days (IL-2 and IL-4) or 2 h (IFN-y and actin). A positive IFN-y
signal was observed with ConA Bi RNA when filters were exposed for
9 h (data not presented). The approximate sizes (in kilobases) of the
indicated cytokine transcripts are indicated on the left of each panel.
served with F. hepatica-specific Th-cell clones G1.1H5 and
G1.1H12 (data not presented). For this reason, total cellular
RNA was harvested from T-cell clones 8 h following ConA
stimulation. A heterogeneous pattern of cytokine expression
by the panel of 17 F. hepatica-specific Th-cell clones was
observed, with the majority of clones expressing an unre-
stricted (ThO) or intermediate cytokine profile (Fig. 2). Six
clones expressed all three cytokine mRNAs (G8.2G10,
G8.3B7, G1.3E11, Gl.lH12, G8.2B1A, and G8.2D9), three
clones expressed IL-2 and IL-4 mRNAs with barely detectable
IFN-y mRNA (G8.2C12, G8.2C1, and G8.2C3), and two
clones expressed IL-4 and IFN--y mRNAs with barely detect-
able IL-2 mRNA (G1.1H5 and G1.3F7). The remaining six
clones exhibited relatively strong expression of IL-4 mRNA
with little or no IL-2 or IFN--y mRNA (G1.3G10, G1.3G4,
G1.3B11, G1.2H4, G1.3G5, and G1.2B6), a pattern character-
istic of the Th2 cytokine profile described for murine (35) and
human (43) T cells. mRNAs obtained from clones Gl.1H5 and
G1.3G10 following stimulation with F. hepatica antigen and
irradiated PBMC as a source of APC exhibited cytokine
profiles identical to those observed following ConA stimula-
tion (data not presented).
824 BROWN ET AL. INFEC-F. IMMUN.

Determinant

CD4 CD8 CD45R L-Selectin

ThO Clone
GLIH5

Gl.IH12
1t __NL 1^LI-
G8.2C 12

G8.2G
Th2 Clone
G 1.2H4

GI.3G1() IL 11 ______ o 0
Lo

G 1.3G4 E
z
G 1.2B6 ._m
_b
en
PBMC
x
GI

G8

Log Fluorescence Intensity

FIG. 3. Comparison of the expression of CD4, CD8, CD45R, and L selectin on F. hepatica-specific Th-cell clones and autologous PBMC. T-cell
clones or PBMC, indicated on the left, were stained with MAb directed at the cell surface determinants indicated at the top of the figure. Data
are presented for 10,000 cells stained with MAb and fluorescein isothiocyanate goat anti-mouse immunoglobulin. Histograms show the log
fluorescence intensity (abscissa) and the relative number of positive cells (ordinate). Negative profiles, for which only the secondary antibody was
used, are indicated by the shaded areas of the curve.

Surface expression of CD45 isoforms and L selectin. To
determine whether functional differences in the Th-cell clones,
characterized by the differential expression of cytokines, cor-
related with differences in surface phenotype, the Th-cell
clones were examined for expression of high- and low-molec-
ular-weight isoforms of the common leukocyte surface antigen
CD45 and the peripheral lymph node homing receptor L
selectin. Figure 3 compares the surface expression of CD4,
CD8, CD45R, and L selectin on PBMC and on Th-cell clones
selected for differences in cytokine mRNA expression. All
Th-cell clones derived from both cattle expressed low levels of
the high-molecular-weight isoform of CD45 (CD45R), which is
expressed on naive T cells in cattle (26). In contrast, 71 and
80% of PBMC from animals Gl and G8, respectively, ex-
pressed the CD45R isoform. In addition, all Th-cell clones of
animal G8 expressed high levels of the low-molecular-weight
isoform of CD45, designated CD45RO, which is a polymorphic
determinant present on freshly isolated memory T cells (32,
33). Thirty-six percent of PBMC from animal G8 expressed
this determinant, whereas leukocytes from animal Gl did not
express the CD45RO determinant identified by MAb ILA-150.
L selectin was expressed on PBMC and on all T-cell clones
examined.
DISCUSSION
The basis for acquired resistance to the cattle liver fluke F.
hepatica has not been defined, but it is likely that both humoral
and cell-mediated responses are important for acquired immu-
nity. Through the elaboration of cytokines, T cells play a
VOL. 62, 1994 F. HEPATICA-SPECIFIC ThO AND Th2 CELLS IN
central role in immunity to the related parasite S. mansoni,
acting as either helper cells for antibody production or as
inflammatory cells that participate in the events leading to
activation of larvicidal macrophages (11, 28, 48). Furthermore,
through the production of important immunoregulatory cyto-
kines, including IL-10, T cells may modulate a protective host
response. Therefore, as a first step in unraveling the complex
cellular interactions leading to resistance in bovine fascioliasis,
we have performed studies designed to characterize the T-cell
response against F. hepatica. Cattle Gl and G8 used for
experimental infections were genetically identical to animals
G3 and G6, respectively, which were infected with B. bovis and
used as a source of B. bovis-specific Th-cell clones described in
a related study (7).
Between 5 and 7 months following oral infection with F.
hepatica metacercariae, PBMC from cattle Gl and G8 were
tested for parasite-specific proliferation. As reported by others
(37), F. hepatica-specific responses were not detected during
this time (data not presented). However, chronic or recent
infection was indicated by the presence of F. hepatica-specific
antibodies in serum sampled from the cattle from 3 to 10
months following infection, which, in the case of animal Gl,
detected a previously unrecognized band on a Western blot at
10 months postinfection.
To overcome the problems associated with in vitro micro-
proliferation assays performed with polyclonal PBMC (49),
short-term T-cell lines were established and cloned by limiting
the dilution. Unlike PBMC, T-cell lines did proliferate specif-
ically to antigen as early as 1 week after their initiation and
exhibited strong antigen-specific responses for an additional 2
to 3 weeks of culture. However, many of the cell lines could not
be maintained in culture for more than 3 or 4 weeks, and two
cell lines maintained for 7 weeks displayed reduced levels of
proliferation. Similar observations were made with B. bovis-
specific T-cell lines (2, 5), in which antigen-specific prolifera-
tion ceased by 11 to 15 weeks of culture. In the studies
performed with B. bovis, decreased antigen responsiveness
correlated with the disappearance of CD4+ T cells and the
predominance of yl8 T cells in the cell lines (2). y18 T cells
were also identified in the cell lines described in the present
study that were stimulated with F. hepatica adult worm antigen,
reaching levels of approximately 50% by 6 weeks of culture,
coincident with a decline in F. hepatica-specific proliferation.
Depletion of the majority of /y8 T cells by antibody- and
complement-mediated cell lysis did not prevent the ability of
the surviving cells to respond to antigen and in fact often
resulted in enhanced proliferative responses. Furthermore,
when two cell lines that were not depleted of /y8 T cells were
cloned after 3 weeks, only ryl8 T-cell clones were obtained, and
these clones did not proliferate in response to F. hepatica. In
contrast, when T-cell lines depleted of the majority of -y/8
TcR+ T cells were cloned, 22 F. hepatica-specific CD4+ T-cell
clones were obtained, whereas no CD8+ T-cell clones and only
one y/8 T-cell clone were obtained (data not presented). T-cell
lines containing fewer than 10% CD8+ T cells were also
capable of vigorous antigen-specific proliferation. Collectively,
these observations indicate that adult worm extract of F.
hepatica preferentially stimulates the proliferation of CD4+ T
cells in vitro and, although not formally proven, suggest that
the presence of -y/8 T cells may impede the ability of CD4+ T
cells to proliferate to F. hepatica, as shown for B. bovis (2).
A spectrum of cytokine mRNA profiles was expressed by the
F. hepatica-specific Th-cell clones, ranging from a Th2 profile,
consisting of strong IL-4 with weak or undetectable IL-2 and
IFN-y mRNA expression, to an unrestricted pattern of all
three cytokine mRNAs. Intermediate patterns of cytokines
CATTLE 825
were also observed, including combinations of IL-2 and IL-4
but no IFN-y, and IL-4 and IFN--y but no IL-2. The CD8+
IL-2-dependent cell line C99.G1.G3 expresses IL-4 as well as
IL-2 mRNA upon activation; however, we cannot presently
determine whether the cell line actually responds to IL-4. For
this reason, it is difficult to correlate IL-2 or IL-4 secretion with
mRNA expression. However, F. hepatica-specific Th-cell
clones that expressed high levels of IL-4 mRNA and recipro-
cally low levels of IL-2 mRNA (i.e., clones G1.3G10, G1.3G4,
G1.3B11, G1.1H5, G1.2H4, G1.3G5, G1.3F7, and G1.2B6)
produced only 0 to 2 U of IL-2 or IL-4 activity per ml, whereas
clones with moderate IL-2 and low IL-4 mRNA expression
(i.e., clones G8.2C1, G8.2C3, and G8.2D9) produced 26 U of
IL-2 or IL-4 activity per ml upon ConA stimulation. These
results indicate that cell line C99.G1.G3 responds poorly, if at
all, to IL-4. IFN-y titers in the culture supernatants correlated,
in most cases, with the level of IFN--y mRNA expressed by
these clones. However, higher titers of IFN--y were detected in
the supernatants of clones G1.3B11 and G1.3G5 than would be
anticipated on the basis of mRNA expression. The reason for
this discrepancy is unclear; however, in studies performed with
murine Th-cell clones, a lack of positive correlation between
IL-4 and IFN--y activities and mRNA expression was often
observed as well (31).
Previous studies in our laboratory also revealed a heteroge-
neity in cytokine mRNA expression by a panel of Th-cell
clones specific for B. bovis, whereby the majority of Th-cell
clones expressed various combinations of cytokines that did
not fit into classical Thl or Th2 profiles (7). In contrast to the
results presented here, however, no Th2-cell clones specific for
B. bovis were detected, whereas several Thl-cell clones were
identified (7, 9). The use of ConA as a stimulus for cytokine
induction does not explain the relative abundance of clones
with unrestricted cytokine profiles, since this mitogen activates
T cells through triggering of the TcR-CD4 complex in the same
way that antigen does and results in qualitatively similar
cytokine expression (21, 29, 35, 44). F. hepatica-specific ThO-
and Th2-like cell clones also had similar cytokine profiles
following stimulation with either ConA or antigen and APC.
The predominance of T-cell clones expressing neither Thl nor
Th2 cytokine profiles may reflect the fact that the cells were
analyzed after being in culture for relatively short periods of
time (i.e., several weeks). Similar unrestricted cytokine pat-
terns were observed with short-term Th-cell clones from
humans and mice (21, 31, 34, 39, 45, 50, 52), whereas the
original description of restricted Thl and Th2 cytokine profiles
was based on analysis of a panel of murine T-cell clones in
culture for months or years (35, 36). In vitro selection of either
Thl- or Th2-like cells from cells expressing unrestricted cyto-
kines was reported to occur after several months of culture
(36).
An alternative explanation for the relative abundance of
clones with unrestricted cytokine profiles may be the nature of
antigen used for in vitro stimulation. In experiments per-
formed with both B. bovis and F. hepatica, we employed
unfractionated parasite extracts that could potentially stimu-
late numerous clones of T cells with specificity for distinct
antigens, leading to simultaneous production of IL-4 and
IFN--y in the cultures. The presence of immunoregulatory
cytokines IL-4 and IFN--y in bulk cultures has been shown to
influence the profile of cytokines subsequently expressed by T
cells cloned from the population (34, 51), resulting in Th-cell
clones that do not fit into reciprocal Thl- and Th2-cell subsets.
In support of this possibility, very high levels of IL-2, IL-4, and
IFN-,y mRNA were detected in polyclonal T cells, obtained
from PBMC of infected cattle Gl and G8, that were stimulated
826 BROWN ET AL.
for 6 days with F. hepatica and then 8 h with ConA (data not
presented).
A comparison of IFN--y titers in the supernatants of Th cell
clones specific for B. bovis (7, 8) with those of Th-cell clones
specific for F. hepatica revealed that the latter set of clones
secreted relatively low levels of IFN-y. Only 1 (6%) of the 17
F. hepatica-specific clones produced more than 128 U of IFN--y
per ml, whereas 12 (57%) of the 21 B. bovis-specific Th-cell
clones, which were generated and tested under the same
culture conditions, secreted between 200 and 800 U of IFN--y
per ml. In fact, 6 of 8 Th-cell clones that secreted >200 U of
IFN--y per ml were derived from cattle G3 and G6 (7), the
genetically identical siblings of cattle Gl and G8. These
findings indicate that differences in the levels of IFN--y pro-
duced by the Th-cell clones generated from cattle infected with
the different parasites cannot be solely explained by host
genetic differences and may be related to the parasite infection
itself.
As anticipated, all of the Th-cell clones expressed a memory
T-cell surface phenotype (CD45RIW and, on G8 clones,
CD45ROhEh), and there were no apparent differences in
expression of CD45R on T-cell clones with unrestricted or
Th2-like cytokine profiles. The Th-cell clones also expressed L
selectin. We similarly showed no differences in expression of
either CD45R or L selectin on ThO- or Thl-cell clones specific
for B. bovis (7). Thus, Th cells displaying different cytokine
profiles could not be distinguished by the differential expres-
sion of either CD45 isoforms detected by available MAb or the
lymph node homing receptor L selectin. The relationship
between a Th-cell subtype, defined by cytokine production in
vitro, and the state of cell differentiation leading from naive T
cells to memory T cells in vivo is not clearly defined (36, 50,
51). Furthermore, the events that drive a T cell down a Thl or
Th2 cell pathway are not completely understood. However,
recent evidence supports the theory that naive CD4+ T cells
specific for a given antigenic epitope are not precommitted to
differentiate into either Thl or Th2 cells, but that differentia-
tion into one subtype or the other is influenced by the
environment (APC), where antigen is encountered in vivo (27,
42).
In summary, T-cell clones derived from cattle infected with
F. hepatica expressed Th2 as well as less-restricted Th cytokine
profiles. These data show that helminth antigens can induce
Th2-like T cells in cattle, as described previously for mice (20,
50) and humans (12, 17), but also extend our earlier observa-
tion that the majority of parasite-specific Th-cell clones do not
fit into reciprocal Thl and Th2 cell subtypes (7). Nevertheless,
the differential expression of IFN--y by Th-cell clones specific
for F. hepatica or B. bovis is consistent with a differential
induction of parasite-specific Th cells in response to infection
with helminth or protozoan parasites. It may be that the
relative amounts of the different cytokines produced is more
important in determining the function of a given Th cell than
the absolute presence or absence of a given cytokine (16, 53).
The role of the functionally different subsets of Th cells in
resistance to F. hepatica remains to be discovered. Preliminary
results suggest that IL-10 is expressed by several F. hepatica-
specific Th clones (4), and experiments are planned to deter-
mine the effects of IL-10 on antigen-driven T-cell proliferation.
Studies are also planned to compare Th-cell clones that
express different patterns of cytokines for the capacity to act as
helper cells for F. hepatica-specific B cells. Specifically, induc-
tion of different immunoglobulin subclasses (IgGl, IgG2, IgA,
and IgE) will be examined. These and additional T-cell clones
can also be used to identify adult worm proteins that elicit
INFEcr. IMMUN.
T-cell responses in cattle resistant to challenge infection, as a
means of targeting potential vaccine antigens for this parasite.
ACKNOWLEDGMENTS
We would like to thank Barbara Doughty for infecting the two cattle
used in this study with F. hepatica metacercariae; Chris Howard and
Niall MacHugh (ILRAD) for providing MAbs; Roger Smith and Betty
Rosenbaum for performing flow cytometry; and Kathleen Logan,
Vivienne Woods, and Emily Francis for expert technical assistance.
This research was supported by U.S. Department of Agriculture
CRGO grant 90-37266-5623 (A.C.R.-F.) and NRICGP grant 91-
37206-6821 (W.C.B.).
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